Although there exists extensive information indicating that membrane alterations are involved in the neoplastic process, very little is known concerning the role of phospholipid and sterol alterations in neoplasia, especially in the formation of metastasis. The aim of this research is to investigate in depth the structure and functions of metastatic cell membranes. The role of membrane bilayer lipid asymmetry utilizing tumorigenic cell lines in culture (LM cell variants and B-16 melanoma variants) will be extended to study the same cell lines growing as primary tumors and as metastatic tumors in mice. We recently have established an in vivo model for a line of mouse fibroblasts, LM cells, that grow in vitro in chemically defined serum-free media. Inoculation of LM fibroblasts subcutaneously into athymic (nude) mice produces malignant fibrosarcomas that metastasize to lungs. Membrane characteristics of the metastases such as Na+,K+-ATPase, benzodiazepine receptor binding, phase transitions, and fluidity gradients were markedly differentfrom those of primary tumor membranes. These differences were maintained in culture. The plasma membrane cholesterol/phospholipid ratio was significantly lower in all metastatic cell lines as compared to primary tumor cells. In addition, high metastatic B16-F10 melanoma cells had a lower plasma membrane cholesterol/phospholipid ratio than the low metastatic B16-F1 melanoma cell line. The structure of the B16-F10 melanoma plasma membrane, microsomes, and mitochondria differed markedly from that of the B16-F10 cells. The data indicated that the solid lipid domains of B16-F1 cell membranes had less restriction to rotational motion than those of the B16-F10 cells. The limiting anisotropy of fluorescence probes (proportional to "fluidity") was significantly lower in the outer monolayer of B16-F1 melanoma but not B16-F10 melanoma plasma membranes. Transmembrane functions will be evaluated by measuring cell membrane Na+,K+-ATPase. Basic biochemical and biological mechanisms involved with methylation of phospholipid (benzodiazepine receptor) and of transmembrane processes (Na+,K+-ATPase and prostaglandin-mediated adenylate cyclase) will be investigated in detail and correlated with alterations in membrane lipid asymmetry in the metastatic cell membranes as compared to primary tumor cell plasma membranes both in vivo and in culture. Results will be correlated to determine if alterations in membrane lipid asymmetry of several tumorigenic cell lines are associated with enhanced tumorigenicity and metastatic capability in the animal models. (A)